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. 2021 Apr 27;12(5):822–826. doi: 10.1021/acsmedchemlett.1c00111

Turning a Quinoline-based Steroidal Anticancer Agent into Fluorescent Dye for its Tracking by Cell Imaging

René Maltais †, Jenny Roy †, Donald Poirier †,‡,*
PMCID: PMC8155233  PMID: 34055232

Abstract

graphic file with name ml1c00111_0006.jpg

RM-581 is an aminosteroid derivative comprised of a steroid core and a quinoline side chain showing potent cytotoxic activity on several types of cancer cells but for which the mechanism of action (MoA) remains to be fully elucidated. The opportunity to turn RM-581 into a fluorescent probe was explored because the addition of a N-dimethyl group was recently reported to induce fluorescence to quinoline derivatives. After the chemical synthesis of the N-dimethyl analogue of RM-581 (RM-581-Fluo), its fluorescent properties, as well as its cytotoxic activity in breast cancer MCF-7 cells, were confirmed. A cell imaging experiment in MCF-7 cells using confocal microscopy then revealed that RM-581-Fluo accumulated into the endoplasmic reticulum (ER) as highlighted by its colocalization with an ER-Tracker dye. This work provides a new tool for RM-581 MoA investigations as well as being a relevant example of a tailor-made quinolone-fluorescent version of a bioactive molecule.

Keywords: Fluorophore, cell imaging, quinoline, steroid, anticancer agent, breast cancer

Deciphering the mechanism of action (MoA) of a bioactive molecule identified from a phenotypic screening approach is a key step to support its further pharmaceutical development into a drug.1−3 Cell imaging using a fluorescent version of a bioactive molecule is an enabling technology toward this end.4,5 However, the nature and size of the labeling fluorophore attached to a small molecule often interfere with its ability to bind to the biological target of interest, thus limiting the validity of inherent cell imaging.6−8 Ideally, the fluorophore used should be as small as possible to most realistically reflect the original unlabeled molecule’s behavior.9 This is particularly true in the case of a lead compound identified from a phenotypic approach when its related biological target is unknown, and thus the assumption that the fluorophore should not interfere with this target cannot be virtually estimated.

Aminosteroid derivatives are promising pro-apoptotic small molecules initially identified from a phenotypic screening approach.10,11 RM-581 (Figure 1) is the lead candidate emerging from this family and shows selective cytotoxicity in several cancer models (in vitro and in vivo) but whose MoA is not yet fully understood.12,13 Even though endoplasmic reticulum (ER)-stress apoptosis was noted behind aminosteroid derivative anticancer activity,13,14 a deeper MoA investigation is required to support its translation toward clinical trials. Recently, our attention was focused on the work of Jun et al., who showed the fluorescence potential of dimethylamino-quinoline derivatives.15 Having a quinoline pharmacophore on aminosteroid RM-581, we were interested in turning our anticancer agent into a fluorescent one (RM-581-Fluo; Figure 1) by adding a small dimethyl amino group and using this opportunity to provide a new tool for RM-581 MoA studies. Also, considering the large number of quinoline-based anticancer agents,16−23 a successful result could offer a relevant example for the use of such a fluorophore to support biological studies related to this class of pharmaceutical compounds.

Figure 1.

Figure 1

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A fluorescent version of RM-581 (RM-581-Fluo) was obtained by adding a small dimethylamino (N-dimethyl) group at position 7 of its quinoline side chain.

RM-581-Fluo was prepared following a seven-step chemical synthesis (Scheme 1) starting from commercially available estrone (E1). The key intermediate 1 was first obtained in four steps from E1, as previously reported.12 Coupling of Fmoc-l-proline with 2-piperazino-steroid 1 followed by the deprotection of Fmoc with TBAF in DMF24 leads to free proline derivative 2. Using HBTU as a coupling agent, compound 2 was then reacted with 7-(dimethylamino)quinoline-2-carboxylic acid, which was prepared from methyl 7-bromoquinoline-2-carboxylate using a procedure reported by Ueki and Amemiya25 to give RM-581-Fluo as a yellow fluorescent amorphous solid. When compared to RM-581, the NMR spectra of RM-581-Fluo are identical except for the signals associated with the presence of a dimethylamino group on the quinoline nucleus (especially two additional signals at 3.13 and 40.0 ppm in 1H and 13C NMR, respectively). As previously observed for aminosteroids RM-133 and RM-581,12,26 two rotamers were seen in NMR spectra of RM-581-Fluo (Supporting Information (SI), Figures S1–S3 and Table S1). In fact, this splitting of certain protons and carbons in NMR occurs only when the proline nitrogen is substituted by a carbonyl derivative (amide) such as for compound 2 and RM-581-Fluo.

Scheme 1.

Scheme 1

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Reagents and conditions: (a) Fmoc-proline, HBTU, DIPEA, DMF, rt (yield 53%); (b) TBAF, DMF, rt (yield 37%); (c) sodium 7-(dimethylamino)quinoline-2-carboxylate, HBTU, DIPEA, DMF, rt (yield 58%).

Once compound RM-581-Fluo was synthesized and characterized, it was tested in breast cancer MCF-7 cells to verify its ability to block cell proliferation. As results, the RM-581-Fluo EC50 value (7.1 μM) was found to be close to the RM-581 value (EC50 = 3.7 μM), with only a slight (1.9-fold) loss of activity (Figure 2). Considering their similar molecular structure, physicochemical and ADME properties (SI, Tables S2–S3), as well as cytotoxic activity, RM-581-Fluo seems to be a valuable probe of RM-581 that was further tested for its fluorescent properties.

Figure 2.

Figure 2

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Comparison of the effective concentrations reducing cell growth (EC50 values) of RM-581-Fluo vs RM-581. MCF-7 cells were treated 3 days with different concentrations of each aminosteroid. EC50 = 7.1 and 3.7 μM for RM-581-Fluo and RM-581, respectively.

The fluorescence spectra of RM-581-Fluo was determined at a concentration of 50 μM, first in phosphate buffered saline (PBS) and next in the same culture medium as used for our cancer cell proliferation and imaging assays. In PBS, the maximum excitation (major peak amplitude) of RM-581-Fluo was observed at 358 nm, the maximal emission at this wavelength was 447 nm, and no difference was observed at the two pH levels tested of 7.4 and 4.0 (SI, Figures S4–S5). In cell culture medium, the maximum excitation (major peak amplitude) of RM-581 was observed at 365 nm, a value that is close to 358 nm in PBS. When excited at 365 nm, a biphasic pattern of maximum emission (two peaks at 448 and 505 nm) was obtained at a pH of 7.3 (Figure 3A). Because the addition of a N-dimethyl group at position 7 of quinoline has been reported to be a fluorescent tunable scaffold in acidic pH,15 we also verified the emission spectra at a pH of 4.0 (Figure 3A). At this pH level, we observed an emission peak at 454 nm and a smaller one (shoulder) at 505 nm. We also measured the emission when excited at 470 nm to see the potential of RM-581-Fluo to emit in green color at pH levels of 7.3 and 4.0 (Figure 3B). The intensity of fluorescence obtained at this excitation wavelength was, however, lower than that observed at 365 nm. In addition to 358, 365, and 470 nm, two other excitation wavelengths (395 and 531 nm) were also tested. No emission was observed when RM-581-Fluo was excited at 531 nm, but when excited at 395 nm, a biphasic pattern of maximum emission (2 peaks at 448 and 505 nm) very similar to that observed for the excitation at 365 nm was obtained (SI, Figure S6).

Figure 3.

Figure 3

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Emission spectra of RM-581-Fluo (50 μM) in DMEM-F12 cell culture medium as measured with a spectrophotometer. (A) pH 7.3 (λex = 365 nm, λem max = 448 and 505 nm) and pH 4.0 (λex = 365 nm, λem max = 454 nm). (B) pH 7.3 (λex = 470 nm, λem max = 525 nm) and pH 4.0 (λex = 470 nm, λem max = 525 nm).

Previous investigations on the MoA of RM-581 anticancer action revealed an induction of ER stress, triggering an unfold protein response (UPR) and subsequent related apoptosis.13 However, no direct observation of RM-581 intracellular localization has been mentioned to date. RM-581-Fluo now offers a unique opportunity to provide direct proof of the RM-581 site of action. Interestingly, RM-581 has been previously reported to be active in breast cancer MCF-7 cells and also in MCF-7 cell-derived xenografts in nude mice, showing complete tumor regression after 28 days of treatment.12 MCF-7 cells were thus selected as a representative cell line to validate the imaging capability of RM-581-Fluo in cancer cells.

Assisted by wide field fluorescence microscopy, we engage cell-imaging experiments, hoping to visualize the penetration of RM-581-Fluo into MCF-7 cells. On the basis of previous studies on cell imaging with steroid derivatives,27,28 we used 30 μM of compound to treat the cells 4 h prior to imaging. Interestingly, we observed a concentration of the compound near nuclei with a stronger and sharper fluorescent emission observed in the green emission range (λex 470/22, λem 525/50 nm) than in the blue emission range (λex 357/44 nm, λem 447/60 nm) (SI, Figure S7). This observation was quite surprising, considering the greater fluorescence intensity of RM-581-Fluo in the blue emission than in the green (Figure 3), but it could be explained by the fact that the cell culture medium also produces a blue emission (SI, Figure S6).

We also determined the influence of concentration and time of treatment on the optical imaging signal. Concentration of RM-581-Fluo (1, 10, and 30 μM) and time of exposure (1, 2, and 4 h) were both tested (SI, Figure S8). Results showed that the increase in the concentration of RM-581-Fluo correlated with the signal intensity (SI, Figure S8A–C). The optimal image was obtained at 30 μM and clearly shows the association of the aminosteroid with the cells. Also, the optimal visualization of the cell-associated compound was observed at the longest exposure time (4 h), where a stronger signal was obtained (SI, Figure S8D–F).

Among the series of pictures obtained with RM-581-Fluo using wide field fluorescence microscopy, there seems to be a more intense signal around the nucleus. Added to the previous MoA studies showing aminosteroid derivative RM-581 as an ER-stress inducer in a variety of cancer cells,12,13 these imaging experiments also suggest that RM-581-Fluo may be concentrated in the ER or close to the ER. To obtain higher resolution images and to better delineate the area around the nucleus, which is a voluminous organelle in MCF-7 cells,29,30 we used confocal microscopy31 and proceeded to a colocalization experiment with ER and cell membrane dyes (Figure 4A–F, and SI, Figure S9–S10). We used the optimal parameters of time and concentration (2 h, 30 μM) previously determined in the wide field microscopy experiments and chose ER-Tracker (in red) and CellMask (in cyan), which offer complementary colors to the blue and/or green color of RM-581-Fluo (parts A and B of Figure 4, respectively). In the results, ER-Tracker emitted a clear signal in red highlighting the ER around the nucleus (Figure 4C). To be noted, the ER-Tracker seems to interfere with the cell membrane (part C vs part D of Figure 4). This phenomena of a nonspecific labeling with ER-Tracker (red) is known in some specialized cell types.32 Interestingly, most of RM-581-Fluo appears to overlap the location with the ER-Tracker (Figure 4E,F), suggesting that the steroid is concentrated on the ER. This result is consistent with the proposed MoA of RM-581 which involved the ER, suggesting that this compound would be a valuable tailor-made tool toward MoA investigations of RM-581 and/or other aminosteroid derivatives. Furthermore, the selective accumulation of RM-581-Fluo in the ER may also suggest that there is an effect of such aminosteroid derivatives on cholesterol homeostasis players,14 especially knowing the important sensitivity of this organelle in cholesterol synthesis control.33 RM-581-Fluo will thus facilitate future investigations related to the effect of aminosteroid derivatives on lipid homeostasis.

Figure 4.

Figure 4

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Representative pictures from confocal microscopy experiments in MCF-7 cells at 60×. Cells were treated for 2 h with 30 μM of RM-581-Fluo prior to microscopy. (A) Cellular localization of RM-581-Fluo (λex 403 nm, λem 460/50 nm). (B) Cellular localization of RM-581-Fluo (λex 403 nm, λem 525/50 nm). (C) Cellular localization of ER-Tracker in red (λex 561 nm, λem 593/40 nm). (D) Cellular localization of CellMask in cyan (λex 642 nm, λem 700/75 nm). (E) Merged images A, C, and D showing a colocalization in pink of RM-581-Fluo (in blue) and ER-Tracker (in red). (F) Merged images B, C, and D showing a colocalization in yellow of RM-581-Fluo (in green) and ER-Tracker (in red). Additional pictures can be found in SI, Figures S9–S10.

In summary, the addition of a small N-dimethyl group to the quinoline moiety of aminosteroid RM-581 leads to a potent and fluorescent anticancer agent. Obtained through a seven-step chemical synthesis from estrone, RM-581-Fluo did not accumulate in the cell membrane and nucleus but was found to accumulate in the ER. These novel data suggest that RM-581-Fluo may exert its action in ER, and this result is in agreement with previous studies suggesting that aminosteroid derivatives trigger apoptosis by ER-stress aggravation.12−14 This work opens the door to future investigations using complementary fluorescent probes and/or live cell imaging tracking experiments to provide a deeper understanding of aminosteroid derivative MoA. More generally, this work also highlights the potential of the addition of a small N-dimethyl group to turn a bioactive compound bearing a quinoline moiety into a fluorescent compound. Considering the large number of molecules bearing a quinoline pharmacophore, the potential of this imaging approach in the development of quinoline-like drugs is quite promising.

Acknowledgments

We thank Jean-François Thériault (CHU de Québec) for his help during the acquisition of fluorescence spectra, Adel Achouba (INSPQ) for providing HRMS spectra, and Dr. Jean-Philippe Lambert (CHU de Québec) for allowing the use of the wide field fluorescent microscope (EVOS M5000 Imaging System) and the bioimaging platform of the CHU de Québec Research Center for the technical assistance of a spinning disc confocal microscope (Quorum technologies-WaveFX). We are also grateful to Micheline Harvey for careful reading of this manuscript.

Glossary

Abbreviations

ADME

absorption, distribution, metabolism and excretion

DAPI

4′,6-diamidino-2-phenylindole

DCM

dichloromethane

DMF

dimethylformamide

DMSO

dimethyl sulfoxide

EC50

effective concentration reducing the cell growth by 50%

ER

endoplasmic reticulum

FCC

flash column chromatography

Fmoc

fluorenylmethoxycarbonyl

HBTU

2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

MoA

mechanism of action

MTS

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium

PBS

phosphate buffered saline

RuPhos-G2

chloro(2-dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl) [2-(2′-amino-1,1′-biphenyl)]palladium(II)

TBAF

tetrabutylammonium fluoride

UPR

unfolded protein response

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00111.

  • Experimental procedures: Material and methods, chemical synthesis, cell proliferation assay, fluorescent properties of RM-581-Fluo, wide field fluorescent microscopy and confocal microscopy. Assignment of all carbon signals for compounds 1–3 and RM-581-Fluo. Physicochemical and ADME properties of RM-581-Fluo. 1H NMR spectrum, 13C NMR spectrum, and HPLC chromatogram of RM-581-Fluo. Excitation and emission spectra of RM-581-Fluo in PBS at pH 7.3 and at pH 4.0. Emission spectra of RM-581-Fluo in cell culture medium. Wide field fluorescence microscopy picture of MCF-7 cells (RM-581-Fluo vs time and concentration). Confocal imaging pictures of RM-581, ER-Tracker, and CellMask (PDF)

Author Contributions

R.M. and D.P. participated in the writing of the manuscript. R.M. designed the composition of the fluorescent dye and performed the chemical synthesis. J.R. performed in vitro experiments.

Ministère de l’économie et de l’innovation (MÉI) du Québec—Programme de soutien aux organismes de recherche et d’innovation (PSO-2D-validation médicament) and Société de valorisation des applications de la recherche (SOVAR).

The authors declare the following competing financial interest(s): R.M., J.R., and D.P. have ownership interest on patent application and patent related to aminosteroid derivatives.

Supplementary Material

ml1c00111_si_001.pdf (908.2KB, pdf)

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Supplementary Materials

ml1c00111_si_001.pdf (908.2KB, pdf)

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